This application claims the benefit of Provisional Application No. 60/488,945 filed Jul. 21, 2003.
This invention relates to the use of a reagent for automated and manual analysis of nucleated cells in suspension, particularly the analysis and enumeration of residual white blood cells or leukocytes with a high degree of accuracy and precision in leuko-reduced blood products including concentrated platelet and packed red blood cell (“RBC”) products.
Various methods and systems are known for counting or enumerating the amount of residual white blood cells or leukocytes in blood banking products such as red blood cell concentrates, platelet concentrates, and plasma. Methods for counting the number of residual white blood cells in blood banking products include microscopic examination of manually prepared dilutions of these products in specially designed hemocytometers and flow-cytometric enumeration of white blood cells. (See Moroff et al, “Validation of the Use of the Nageotte Hemocytometer to Count Low Levels of White Cells in White Cell-Reduced Platelet Components” TRANSFUSION, vol. 34, pp. 35-38, (1994), AABB Technical Manual. Method 8.8 “Counting Residual White Cells in Leukocyte-Reduced Blood and Components”, Goodfellow et al. “The United Kingdom National External Quality Assessment Scheme Gating and Standardization Strategy for Use in Residual WBC Counting of WBC-Reduced Blood Components”, TRANSFUSION, vol. 42, pp. 738-746, (2002), Vachula et al. “A Flow Cytometric Method for Counting Very Low Levels of White Cells in Blood and Blood Components.” TRANSFUSION vol. 33, pp. 262-267, (1993).
Current methods for the enumeration of leukocytes use relatively high pHs of about 8.5 and above to facilitate the elimination of the interference of other blood cell-types by differentially lysing these other blood cell types, while leaving a residue of white blood cells for identification and enumeration. The disadvantage of operating at pH's of 8.5 and above is that rapid deterioration of the white blood cells occurs.
Current methods also use harsh organic solvents as leuko-protective agent, including ether/alcohols, such as ethoxyethanol, butoxyethanol, ethoxyethoxyethanol, methoxyethanol, ethyl ether and the like; alcohols, such as isopropyl alcohol dimethyl ethylene glycol, diethylethylene glycol and the like; ketones, such as acetone, methyl ethyl ketone and the like; aprotic solvents, such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide and the like; and furans, such as tetrahydrofuran and the like.
U.S. Pat. No. 4,637,986 to Brown. et al. discloses other leuko-protective agents. The use of such organic solvents is disadvantageous because they can damage or have an adverse effect on the plastic components of automated clinical analyzers operating as flow cytometers. Plastic materials are commonly used in the fabrication of numerous components of automated analyzers including reaction chambers, seals, tubing, waste and storage containers and optical compartments. Chemical solvents that attack these components can render the analytical system inoperable.
Harsh organic solvents that are commonly used as leuko-protective agents can adversely affect the strength, flexibility, surface appearance, color and dimensions of plastic components of automated clinical analyzers. Harsh organic solvents can damage the plastic components of automated clinical analyzers by chemical attack on the plastic polymer chain, such as oxidation and depolymerization, and can cause physical damage when the plastic components absorb the harsh solvents that can result in softening and swelling of the plastic component, permeation of the solvent through the plastic component, and dissolution of the plastic component in a solvent.
Harsh organic solvents include ketones such as acetone or methyl ethyl ketone can dissolve polystyrene and adversely affect low density polyethylene (LDPE) and high density polyethylene (HDPE), polypropylene (PP), neoprene rubbers, acrylates such as Plexiglass™, and Viton™. Ethers, such as ethyl and propyl ethers, can cause swelling, distortion or dissolution of polypropylene, LDPE, HDPE, PP and neoprene rubber. Alcohols can also adversely affect polyethylene. Therefore, use of harsh organic solvents should be avoided.
Automated analysis is more precise than current manual methods for the enumeration of relatively small amounts of leukocytes. The current reference manual analysis method involves introducing a leukocyte-reduced blood sample into a Nageotte hemocytometer. This is a glass slide with small grid on the surface. The sample is introduced to the device and examined under a microscope. Using the grid the white blood cells can be counted.
The total volume of a Nageotte hemocytometer chamber enables about 100 uL of diluted sample to be analyzed. This manual method is time consuming and highly dependent upon operator skill and experience.
In contrast, automated analysis can be adapted to analyze higher sample volumes thus obtaining a better statistical sampling and thereby improve reproducibility and precision of the result. Automation also offers the added advantage of eliminating operator error.